Driving electrochemical reactions at the microscale using CMOS microelectrode arrays

Precise control of pH values at electrode interfaces enables the systematic investigation of pH-dependent processes by electrochemical means. In this work, we employed high-density complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs) as miniaturized systems to induce and confine electrochemical reactions in areas corresponding to the pitch of single electrodes (17.5 μm). First, we present a strategy for generating localized pH patterns on the surface of the CMOS MEA with unprecedented spatial resolution. Leveraging the versatile routing capabilities of the switch matrix beneath the CMOS MEA, we created arbitrary combinations of anodic and cathodic electrodes and hence pH patterns. Moreover, we utilized the system to produce polymeric surface patterns by additive and subtractive methods. For additive patterning, we controlled the in situ formation of polydopamine at the microelectrode surface through oxidation of free dopamine above a threshold pH > 8.5. For subtractive patterning, we removed cell-adhesive poly-l-lysine from the electrode surface and backfilled the voids with antifouling polymers. Such polymers were chosen to provide a proof-of-concept application of controlling neuronal growth via electrochemically-induced patterns on the CMOS MEA surface. Importantly, our platform is compatible with commercially available high-density MEAs and requires no custom equipment, rendering the findings generalizable and accessible.


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&XUUHQW>$@ H %XIIHU %XIIHU61$5) Figure S5: Testing the electroactive properties of SNARF using cyclic voltammetry (CV).The measurements were performed using an Autolab potentiostat (PGSTAT302N).A three-electrode system was set up with a platinum wire serving as the working electrode.Another platinum wire served as a counter electrode and an Ag/AgCl electrode as the reference electrode.All electrodes were placed in a well of a 12-well plate, which was filled with either the pure buffer solution or the buffer solution with SNARF added at concentrations mentioned in the "Materials and Methods" section of the main manuscript (55 µM).The potential was cycled three times between 0 and 1.6 V with a speed of 200 mV/s.Here, the last cycle is shown for both cases.No oxidation or reduction peaks appeared after the buffer solution was replaced with the buffer solution containing SNARF (retaining the positions of the 3 electrodes), demonstrating that SNARF is not electroactive under the conditions tested in the manuscript.

Figure S1 :
Figure S1: SEM images of a microelectrode on nonplanar CMOS MEAs with (A) bare platinum and with (B) platinum black-coated electrodes.

Figure S2 :Figure S3 :
FigureS2: Equivalent electrical circuits.The circumferential reference electrode is shown in grey, while microelectrodes are shown in black.Between the reference electrode and the system ground, a voltage V ref is measured, which in the standard case coincides with the mid-potential of the chip.An external voltage is applied via a source meter, providing the voltage V SM U .The grounds of the MEA and the SMU are connected.A In case neighboring electrodes are left floating, the applied voltage between the cathode and the reference electrode (that serves as the anode as it is the second point in the electrochemical circuit with a defined potential) is defined by the difference of V SM U and V ref .Since the anode and cathode are not in close proximity, resulting regions of elevated pH are diffuse as shown in main text Fig.3A/B.B In case neighboring electrodes are connected to a DAC with a defined voltage (V DAC , in our case: mid-potential of the chip) the anode-cathode pair is in direct vicinity to each other and the potential difference is defined by the difference of V SM U and V DAC .This configuration leads to sharp regions of elevated pH as shown in main text Fig.3C/D.

Figure S4 :
Figure S4: Relationship between pH and fluorescent ratio and fit used for pH quantification.Three images on different locations on two PtB CMOS MEAs were acquired and a linear fit was performed using linear regression on the resulting fluorescent ratios.The error bars indicate the standard deviation.

Figure S6 :Figure S7 :
Figure S6: Fluorescence emission of SNARF on three platinum black (PtB) and two platinum (Pt) chips.The fluorescence intensity in the green window decreases with increasing pH.The intensity in the red window increases with increasing pH.PtB electrodes are able to induce larger pH changes.

Figure S8 :Figure S9 :Figure S10 :Figure S11 :
Figure S8: Whole chip image after polydopamine deposition.The labeled voltage was applied for 30 min at each square.When dopamine was present in the solution, dark squares were formed on the FITC-PLL-coated CMOS surface.When only the buffer was present, a hollow square appeared indicating the removal of PLL.

Figure S12 :
Figure S12: Polymerization of hydrogels on the CMOS MEA.A Enzymatically-crosslinked polyethylene glycol (PEG) hydrogel was polymerized on the CMOS MEA for different polymerization times ranging from 20 -35 min.The hydrogel precursor solution consisted of 1.5% PEG dissolved in 0.125 mM HEPES/sodium acetate buffer.Lysine-tagged fluorescein isothiocyanate (Lys-FITC, 10 µM) was added to the precursor solution (1:12.5 v/v ratio) to render the hydrogel fluorescent upon its incorporation into the mesh.The resulting solution was then mixed in a 25:1 v/v ratio with fibrin stabilizing factor XIIIa and the pH was manually adjusted to 5.8.All chemicals were purchased from Ectica Technologies, Switzerland.Post polymerization, the FITC-lysine was found to be attached to the anode.B Primary rat cortical neurons at day in vitro 10 stained with CellTracker Green (CMFDA) dye avoid locations where hydrogels were grown.The extent of the avoidance-zone increases with hydrogel polymerization time.